Figure 1

Characterization of 4T1 engineered cells. (A) Scheme depicting the strategy used to generate our cell models as described in Supplementary Methods. Briefly, 4T1 cells were infected with either a pTZ doxy-inducible lentiviral vector encoding a short hairpin RNA vector for murine IR (shmIR) or a scramble shRNA (shmScr, elsewhere referred to as 4T1/NS). 4T1shmIR cells were then exposed to a second infection with pTZ doxy-inducible lentiviral vector encoding for the human IR-A or IR-B or the corresponding empty vector to generate 4T1shmIR/hIR-A, 4T1shmIR/hIR-B, and 4T1shmIR/EV cells (referred as 4T1/IR-A, 4T/IR-B and 4T1/EV cells, respectively. (B) IR expression in 4T1/NS, 4T1/EV, 4T1/IR-A, or 4T1/IR-B cells was evaluated by Western blot analysis. Cells were grown in 10% FBS in the presence of doxycycline, then lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies to evaluate the expression of both mouse and human IR and β-actin. A representative blot of three independent experiments is shown. The graph on the upper panel represents the mean ± SE of densitometric analysis of three independent experiments, where hIR was normalized over β-actin. (C) IR isoform (IR-A and IR-B) transcripts were obtained from cell clones as indicated in (B). Products of PCR amplification were resolved on a 2.5% agarose gel, and images of PCR products from IR-B (Ex+11, 167 bp) and IR-A (Ex-11, 131 bp) obtained (middle panel). Graphical representation of PCR analysis indicates the percentage of IR-A mRNA calculated as follows: densitometric value of IR-A band/densitometric value of IR-A + IR-B bands (upper panel). Scanning densitometry was performed using ImageJ software. Results are expressed as means ± SE of three independent experiments. (D) Cells (as in A) were analyzed for hIR mRNA expression by qRT-PCR. Values are means ± SE of three separate experiments. (E) Cells were analyzed for mIR mRNA expression by qRT-PCR. 4T1/NS cells were used as control and GAPDH as housekeeping control gene. Values are means ± SE of three separate experiments. (F) 4T1/EV, 4T1/IR-A, and 4T1/IR-B cell monolayers unstimulated or stimulated with insulin at 0.1, 1.0, and 10 nM for 10 min were evaluated for total and phosphorylated IR proteins by Western blot using two different phosphoantibodies, as detailed in Methods. β-actin was used as loading control. The graph panels represent the mean ± SE of densitometric analysis of two independent experiments, where phosphorylated IRs were normalized over β-actin. (G) Cell conditioned medium from 4T1/EV, 4T1/IR-A, and 4T1/IR-B cells or IGF2 at the indicated doses were added to IR-A overexpressing mouse fibroblasts (R-/IR-A). A representative blot of two independent experiments is shown. The graph represents the mean ± SE of densitometric analysis of two independent experiments, where phosphorylated IRs were normalized over β-actin. (H) qRT-PCR measurement of IGF2 mRNA expression in 4T1/EV, 4T1/IR-A, and 4T1/IR-B cell monolayers stimulated or not with insulin 10 nM for 8 h. 3T3-NIH mouse fibroblasts were used as positive control. Values are means ± SE of two separate experiments. (ns, not significant; * p <0.05; ** p <0.01; *** p <0.001; and **** p <0.0001).